We incubated Fusarium semitectum on sorghum and measured the production of zearalenone (ZEN) and ZEN-related compounds (zearalanone (ZAN), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL)) in the culture by LC-MS. Of the five ZEN-related compounds, ZAN and β-ZEL were mainly detected. The concentrations of ZEN and the five ZEN-related compounds increased until 9 days after incubation and then increased slightly or stayed constant between days 9 and 15. The ratios of α-ZEL, β-ZEL, α-ZAL and β-ZAL to ZEN decreased in a similar manner after 7 days, whereas the ratio of ZAN to ZEN remained constant after 5 days. Analysis of naturally contaminated sorghum by LC-MS/MS revealed that the production ratio of α-ZEL to ZEN was inconsistent with that of our in vitro incubation analysis. The results indicate that ZAN might not be suitable for use as an internal standard.